Woon Teck Yap
Section: M1-3, E53-220
Meeting 4
In class Exercise 4
0.8
0.7
0.6
A595
0.5
0.4
0.3
0.2
0.1
0
0
0.5
1
1.5
2
2.5
3
BSA Concentration / 10-2 m g m l-1
Figure 1: Standard curve of absorbance values at 595 nm (A595) against concentration of
bovine serum albumin (BSA), measured in mg ml-1. The standard curve was generated
using the Bio-Rad Bradford Protein Assay in which 200 μl of Bio-Rad dye reagent was
added to 790 μl of column buffer and 10 μl of appropriately diluted BSA stock solution
in a poly-propylene cuvette. BSA stock solution was provided at a concentration of 5 mg
ml-1 and was dilution 1:2, 1:4, 1:8, 1:16 and 1:32, respectively. Absorbances were read at
595 nm in a Milton Roy Spectronic 601 Spectrophotometer.
Woon Teck Yap
Section: M1-3, E53-220
Meeting 4
In class Exercise 4
Table 1: Table showing various characteristics of protein samples obtained at various
steps along the purification pathway. Protein samples analyzed were the crude lysate
(CL), crude lysate supernatant (CL-S), ammonium sulphate precipitation supernantant
(AS-S), ammonium sulphate precipitation pellet (AS-P), DEAE column loading sample
(DEAE-Load), DEAE column eluent (DEAE-Total) and the desalted APTG column
eluent (AF/PD-10). The total protein content and total protein activity of each sample
were analyzed and used to calculate the specific activity, yield and fold purification of the
sample. All figures are reported to 3 significant figures.
Sample
CL
CL-S
AS-S
AS-P
DEAELoad
DEAETotal
AF/
PD-10
Total
Protein/mg
10.4
8.70
8.60
3.75
Total
Activity/U
29300
28600
725
4660
Specific
Activity/U mg-1
2820
3290
84.3
1240
100
97.6
2.47
15.9
Fold
Purification
1.00x
1.16x
0.299x
0.441x
3.68
9030
2450
30.8
0.871x
0.225
470
2090
1.60
0.741x
0.200
306
1530
1.04
0.543x
Yield/%